The present invention relates generally to the aging of proteins resulting from their reaction with glucose and other reducing sugars, and more particularly to the reversing or cleavage of cross-links formed as a consequence of the formation of advanced glycosylation (glycation) end products.
The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food, which was identified by Maillard in 1912. who observed that glucose or other reducing sugars react with amino acids to form adducts that undergo a series of dehydrations and rearrangements to form stable brown pigments. Further studies have suggested that stored and heat treated foods undergo nonenzymatic browning as a result of the reaction between glucose and the polypeptide chain, and that the proteins are resultantly cross-linked and correspondingly exhibit decreased bioavailability. As described in copending application Ser. No. 08/588,249, incorporated herein by reference, these reactions have a parallel in vivo, and have been found to occur with a variety of other body proteins, such as lens crystallins, collagen and nerve proteins. These reactions are accelerated in the presence of elevated glucose levels, as occur in individuals with diabetes mellitus, but still occur in vivo at normal glucose levels. Termed advanced glycosylation (or glycation) end products (AGEs), the cross-linked products involving structural and other proteins within the body leads not only to aberrant physico-chemical properties of, for example, connective tissue, but also results in formation of new chemical structures which are recognized by specific receptors on various cell types and as a consequence of their recognition, initiate pathogenetic mechanisms leading to the complications of diabetes and aging.
Several successful therapeutic approaches have been achieved based upon intervening in the accumulation of AGEs in vivo. One approach, exemplified in U.S. Pat. No. 4,758,583, incorporated herein by reference, concerns the inhibition of the formation of AGEs from its precursors, by the administration of agents such as aminoguanidine and related compounds. By reacting with an early glycosylation product that results from the original reaction between the target protein and glucose, these agents block the formation of AGEs and further formation of AGEs and cross-links in tissues is inhibited. Efficacy of this approach has been demonstrated in numerous animal models of diabetes and aging, including positive effects on macrovascular, renal, retinal, and neural pathology. These data have been reviewed by Vlassara et al., 1994, "Biology of Diseases. Pathogenic effects of advanced glycosylation: biochemical, biologic and clinical implications for diabetes and aging," Laboratory Investigation 70:138-151; Brownlee, 1995, "The pathological implications of protein glycation," Clin. Invest. Med., 18:275-281; and Brownlee, 1995, "Advanced protein glycosylation in diabetes and aging," Ann. Rev. Med. 46:223-34.
In another pharmacological approach to controlling levels of AGEs in tissues, especially in those tissues in which AGE cross-links have already accumulated to levels which are responsible for subclinical or clinical pathology administration of agents that reverse or break AGE cross-links has proven successful. As described in U.S. Pat. No. 5,656,261 and copending U.S. application Ser. Nos. 08/588,249 and 08/848,776, all of which are incorporated herein by reference in their entireties, agents and methods are disclosed which reverse (also termed cleave or break) existing AGE cross-links in vitro and in vivo. Studies demonstrate positive effects of such agents on cardiovascular complications related to aging which are accelerated in experimental diabetes (see Wolffenbuttel et al., 1998, "Breakers of Advanced Glycation End Products Restores Large Artery Properties in Experimental Diabetes," Proc. Nat. Acad. Sci. U.S.A. 95:4630-4634). In these studies, rats diabetic for 9 weeks followed by 1 to 3 weeks administration of an AGE breaker compound resulted in reversal of diabetes-induced increases in large artery stiffness. Parameters that were improved included cardiac output, peripheral resistance, systemic arterial compliance, input impedance of the aorta, and compliance of the carotid artery.
It is toward the identification of additional agents capable of reversing AGE cross-links that the present invention is directed.